Project Summary Influenza remains to be a significant public health problem worldwide, despite the heavy campaign for flu preventative vaccination in many countries. Influenza vaccines must be reformulated annually because of antigenic shift and drift of circulating influenza viral strains. However, reformulated seasonal vaccines do not always match the circulating strains, and there is the ever-present threat that avian influenza viruses may adapt to be transmitted in humans. Another complementary strategy, antiviral therapy, is also widely used for treating influenza infection. There are two classes of antiviral drugs against influenza, neuraminidase (NA) blockers (oseltamivir and zanamivir) and matrix protein 2 (M2) inhibitors (adamantine derivatives), however, all of which face the challenges of evolved drug resistance and simultaneously occurring nonspecific side effects. Therefore, there is an urgent need for developing novel drugs/vaccines and combination therapies against influenza virus infection especially considering that the annual influenza outbreak results in about three to five million cases of severe illness and appropriately 250,000 to 500,000 deaths worldwide. In this research, we hypothesize that a safe therapeutic influenza vaccine can be developed through creation of a new class of live attenuated influenza virus (LAIV) using specific host cell-restricted attenuation. In our preliminary study, using the eight-plasmid reverse genetics, we have constructed a LAIV, which carries an artificial microRNA 93 (amiR-93) expression cassette and expresses a mammalian- specific amiR-93 that can inhibit the expression of a host gene essential for influenza virus replication. Interestingly, a single dose intranasal vaccination with the resultant engineered LAIV provided potent immune protection against challenge with lethal dose of influenza A virus. Additionally, administration of this LAIV 24 hours post lethal influenza infection could completely protect mice against influenza. Based on this feasibility study, we propose to continue evaluating this new class of candidate therapeutic vaccine in both mouse and ferret models of influenza infection. The following two specific aims are proposed: Specific Aim 1: To evaluate the efficacy of a candidate therapeutic influenza vaccine generated by specific host cell-restricted attenuation. Specific Aim 2: To produce and evaluate additional dual host factor-targeted therapeutic vaccine. Our candidate LAIV therapeutic vaccines will be extensively investigated in both mouse and ferret models of influenza infection. We anticipate that the proposed research will identify a novel and safe anti- influenza drug that can also be further developed as a therapeutic vaccine to prevent future influenza re- infection.